MYC is the most frequently amplified oncogene in human cancers occurring in a wide range of tissue types including breast, lung, and prostate. MYC overexpression occurs in at least 30% of all human cancers and frequently correlates with poor clinical outcome and increased chance of relapse. An estimated 450,000 Americans are diagnosed with a MYC-dependent cancer each year. These patients are in need of novel and effective treatment strategies. c-Myc is a helix-loop-helix transcription factor that drives a proliferative cell state by forming a heterodimer with Max, binding sequence-specific DNA elements and stimulating transcription of proliferation-associated genes. Transcription factors are key regulators of cell state as they control the gene expression programs that drive cell type specification and commonly are terminal components of a signaling cascade. These gene expression programs are often deregulated in disease states making transcription factors an ideal class of proteins for therapeutic targeting. However, most transcription factors lack clear pockets for small molecule binding and therefore have been largely considered undruggable with current technologies. A major challenge in the chemical biology field has been to develop potent small molecule inhibitors of transcription factors. While previously published work has identified small molecule inhibitors of c-Myc/Max heterodimerization using truncated proteins in FRET and yeast two-hybrid assays, only a few thousand compounds were screened in each case and the in vitro potency of these inhibitors is limited. Indeed, the potency fails to translate to in vivo activity in animal models. In general, these compounds fail to meet the generally agreed upon criteria for acceptable chemical probes. New chemotypes, which can be successfully developed into chemical probes, are desperately needed.)The proposed research aims to identify inhibitors of c-Myc/Max dimerization and DNA binding using novel technology. A robust high-throughput in vitro assay has been developed to screen for inhibitors of c-Myc/Max dimerization and subsequent binding to its DNA binding site. Secondary biochemical and cellular assays have been developed to validate hits from the primary screen and study them in greater detail. A large high-throughput screen as could be provided through the MLPCN using these robust assays should provide tractable hits for development and validation of biological effect. Medicinal chemistry optimizing these lead molecules through iterative use of downstream assays outlined herein then provides the opportunity to generate useful chemical probes to study c-Myc function. Such probes will hopefully lead the way to new therapeutics against this quintessential cancer target and offer insights into mechanisms for directly inhibiting transcription factors.